The objectives of this proposal are (1) to advance our capability to noninvasively evaluate human normal and pathological corneal mitochondrial function. (2) to investigate the relationship of cellular respiratory function and morphology of endothelial and epithelial cells. The Specific Aims are: (1) to improve the spatial resolution of redox fluorometry to permit measurements on individual cells in basal, superbasal, wing and superficial epithelial cells and endothelial cells, and to test the following hypotheses: (2) that endothelial morphology derives from altered cellular respiration, (3) stressed endothelial cells (transplantation, cataract surgery, Fuchs' dystrophy) have abnormal cellular respiratory function, (4) oxygen concentration in the component layers of the cornea and aqueous humor regulated cellular respiratory function in endothelium and epithelium, (5) oxygen gradients in the rabbit neonate regulate collagen formation in the stroma, (6) reduced oxygen availability at tear film affects mitochondrial function of endothelial cells, and (7) determine the role of intra- and extracellular pH on mitochondrial respiratory function of epithelium and endothelium. The health-relatedness of this project is to further our understanding of how corneal pathology and surgically induced stress alters cellular respiratory function. The ability to perform noninvasive studies on humans will provide early diagnostic indicators of cellular dysfunction. The methods to be used are: intensity measurements of mitochondrial fluorescence, confocal real-time reflectance, and fluorescence measurements of basal epithelium and endothelium, 2-D redox oxygen concentration using phosphorescence lifetimes of porphyrin probes of pH probes, digital image processing to enhance and quantify specular, mitochondrial respiratory function, oxygen gradients, pH gradients, and cellular morphology in normal and stressed corneas.